Identification and functional validation of intracellular protein partners of phosphorothioate-splice switching oligonucleotides through AP-MS

Despite growing interest in therapeutic antisense oligonucleotides (ASOs), the mechanisms governing their intracellular trafficking remain poorly understood. This study investigates the intracellular protein partners of splice-switching oligonucleotides (SSOs) modified with phosphorothioate (PS) backbones of three different chemistries: tricycloDNA (tcDNA), locked nucleic acid (LNA), and 2'-O-methoxyethyl (2′MOE). The SSO studied here targets the exon 51 of the dystrophin pre-mRNA (SSOEx51), as part of an exon skipping strategy for Duchenne muscular dystrophy (DMD). Pulldowns were performed using lysates from differentiated human muscle cells carrying a deletion in the exon 52 of the DMD gene (KM571). Biotinylated SSOs were immobilized on streptavidin-coated magnetic beads, and interacting proteins were captured and identified by nanoLC-MS/MS. In addition to analyzing the effect of different chemistries of ASOs on protein binding, we also evaluated the impact of sequence variation by comparing two additional tcDNA chemistry sequences (named TSB-T3 and TSB-C1). Finally, to enable enrichment analyses, we performed a characterization of the whole proteome of KM571 cells.